Injectable calcium-phosphate cement releasing a bone resorption inhibitor

ABSTRACT

The present invention relates to a macroporous, resorbable and injectable apatitic calcium-phosphate cement with a high compressive strength useful as bone cement and releasing a bone resorption inhibitor, preparation method and uses thereof.

The invention relates to a macroporous, resorbable and injectableapatitic calcium-phosphate cement with a high compressive strengthuseful as bone cement and releasing a bone resorption inhibitor.

Bone is a composite of biopolymers, principally collagen, and aninorganic component identified as carbonate hydroxyapatite, approximatedas (Ca,Mg,Na,M)₁₀(PO₄,CO₃,HPO₄)₆(OH,Cl)₂.

Deregulation of the bone activity of an individual is the cause of manybone pathologies such as osteoporosis, Paget's disease or osteolytictumors. Taking into account, in particular, the increase in human lifeexpectancy, osteoporosis has become a public health problem and muchresearch has been undertaken to remedy it. Since the bone pathologiesunder consideration are caused by an imbalance in bone remodeling to thebenefit of the activity of the osteoclasts, one of the routes oftreatment envisioned consisted in reducing the activity of theosteoclasts, in order to slow down the degradation of the bone material.

Studies performed on various gem-bisphosphonic acids have shown theirinhibitory power on osteoclast activity (G. A. Rodan et al., TherapeuticApproaches to Bone Diseases, 1 Sep. 2000, Vol. 289, Science, pp.1508-1514). The use of some of them as medicaments, especiallyetidronate, clodronate, pamidronate, alendronate, risedronate,tiludronate and ibandronate, has been approved in various countries.Data have been published for other gem-bisphosphonic acid compounds,especially zoledronate, incadronate, olpadronate and neridronate. Thegem-bisphosphonic acids that are used at the present time for thetreatment of bone lesions are used systemically and, as a result, giverise to a few undesirable side effects. They can cause renal disordersand jaw osteonecrosis (Eckert A. W., Cancer Treatment Reviews, 2006, inthe press) when they are administered intravenously, and digestivesystem-disorders, especially oesophagi or stomach ulcers, when they areadministered orally [(Lin J. H., Bone 1996; 18; 75-85) or (Thiébauld D.et al., Osteoporos Int. 1994; 76-73)]. Another drawback of the oraladministration lies in the low level of absorption of the activeprinciple onto bone material.

To date, a wide variety of implant materials have been used to repair,restore, and augment bone. The most commonly used implants includeautologous bone, synthetic polymers and inert metals. Protocols usingthese materials have significant disadvantages that can include patientpain, risk of infection during operations, lack of biocompatibility,cost, and the risk that the inserted hardware can further damage thebone. Therefore, a major goal of biomaterial scientists has been todevelop novel bone substitutes that can be used as alternatives to theseconventional techniques for skeletal repair.

Bone cements, such as cements based on polymethylmethacrylate (PMMA)offer certain advantages in avoiding the use of solid implants, but alsohave several disadvantages. Methacrylates and methacrylic acid are knownirritants to living tissues, and when PMMA-based cements are cured invivo, free radicals are generated, which can damage surrounding tissues.Moreover, the polymerization reaction for these materials is highlyexothermic, and the heat evolved during curing can damage tissues. Inaddition, these materials are not biodegradable.

The concept and potential advantages of an apatitic or calcium phosphatecement (CPC) as a possible restorative material was first introduced byLeGeros et al in 1982 (“Apatitic Calcium Phosphates: PossibleRestorative Materials”, J Dent Res 61(Spec Iss):343).

There are presently several CPC commercial products. CPC have thefollowing advantages: malleability allowing them to adapt to thedefect's site and shape. The introduction of injectable calciumphosphate cements greatly improved the handling and delivery of thecements and opened up areas of new applications for the CPC.

CPC systems consist of a powder and a liquid component. The powdercomponent is usually made up of one or more calcium phosphate compoundswith or without additional calcium salts. Other additives are includedin small amounts to adjust setting times, increase injectability, reducecohesion or swelling time, and/or introduce macroporosity.

Such materials are disclosed, for example, in EP 0 416 761, U.S. Pat.No. 4,880,610, U.S. Pat. No. 5,053,212, EP 0 664 133, EP 0 543 765, WO96/36562, and WO 2004/000374.

FR-2715853 describes compositions for biomaterials forresorption/substitution of support tissues, comprising a mineral phasecomposed of BCP or calcium-titanium-phosphate, and a liquid aqueousphase comprising an aqueous solution of a cellulose-based polymer. Theseinjectable compositions contain no active principle.

Noninjectable bone substitutes, which are in the form of implants, arealso known. For example, H. Denissen et al. (J. Periodontal, Vol. 71,No. 2, February 2000, pp. 280-296) describes implants of hydroxyapatitemodified by absorption of a particular gem-bisphosphonic acid, namely(3-dimethylamino-1-hydroxypropylidene)-1,1-bisphosphonic acid, orolpadronate. The in situ release of the acid is said to promote bonereconstruction. However, hydroxyapatite itself has the drawback of beingvery poorly resorbable.

The international application WO03/074098 describes a modifiedphosphocalcic compound obtained by the addition of a gem-bisphosphonicacid or an alkali metal or alkaline-earth metal salt thereof to asuspension of a precursor phosphocalcic compound in ultrapure water, bystirring the reaction medium at room temperature, and then recoveringthe pellet by centrifugation, washing the pellet with ultrapure water,followed by filtering and drying in air at room temperature. WO03/074098also describes a suspension of the so obtained modified phosphocalciccompound in a solution or an hydrogel and the use of the so obtainedinjectable composition for the treatment of osteoporosis and osteolytictumors. The inventors of WO03/074098 thus recommend a localadministration of a gem-bisphosphonic acid, the phosphocalcic phaseproviding a source of calcium and of phosphate required for stimulationof the bone remodelling.

The present applicant has now surprisingly found a method of producing acalcium-phosphate bone cement i.e. an auto-hardening cement, having acompressive strength close to bone, being resorbable for itssubstitution by new bone material and presenting a release of agem-bisphosphonic compound, i.e. a bisphosphonic acid or a salt thereof,which allows a regulation of bone remodelling.

First, the present inventors have shown that it is possible to obtain anauto-hardening calcium-phosphate cement comprising a gem-bisphosphoniccompound with a setting time suitable for a chirurgical use while saidgem-bisphosphonic compound shows a setting retarder activity. Indeed,the phosphonate groups of the gem-bisphosphonic compounds compete withphosphate groups of the cement paste in the following setting reaction.Consequently, the final product, its setting time and hardness aremodified.

Mechanism Proposal

In presence of the liquid phase, the initial calcium phosphate compoundis partially hydrolysed. Ca²⁺ and PO₄ ²⁻ ions are released.Bisphosphonic compounds chelate to Ca²⁺ ions and may hamper the apatiteprecipitation (see reaction below):

α-Ca₃(PO₄)₂(α-TCP)+H₂O→Ca²⁺+PO₄ ²⁻+(Ca,Na)₁₀(PO₄,HPO₄)₆(OH)₂(CDA)

Second, the present inventors have shown that it is possible to obtain aresorbable calcium-phosphate cement comprising a gem-bisphosphoniccompound. This is an unexpected technical effect since the introductionof a gem-bisphosphonic compound was thought to reduce the resorbabilitypotential of phosphocalcic compounds in general.

Third, the present inventors have shown that it is possible to obtain acalcium-phosphate cement releasing a gem-bisphosphonic compound which isthus able to have its inhibitor activity locally on the osteoclasts.This allows solving the secondary effects of an oral administration.Moreover, the dosage used in the cement (for example: 4 mg/implantedsite) is much lower than the dosage used for an oral administration(10-70 mg/day during several months). Furthermore, it has been shown(Clin Cancer Res. 2006 15; 12(20 Pt 2):6222s-6230s, ChemMedChem. 2006February; 1(2):267-73) that bone has a very high affinity forgem-bisphosphonic compounds. Thus, the gem-bisphosphonic compoundsreleased by the cement according to the invention will be immediatelyabsorbed by bone close around the implantation site and all the dosereleased will be trapped.

DEFINITIONS

A “cement” is the result of the setting of a paste resulting from themixing of a pulverulent solid phase and a liquid phase.

The “setting” of a cement means the hand-off auto-hardening at room orbody temperature of the paste resulting from the mixing of the solidphase and the liquid phase.

An “injectable cement” means a cement paste sufficiently fluid to flowthrough a needle with a diameter of a few millimetres, preferablybetween 1 and 5 mm.

A “calcium phosphate cement” is a cement wherein the pulverulent solidphase is made of a calcium phosphate compound or a mixture of calciumand/or phosphate compounds.

An “apatitic” calcium phosphate cement crystallises in the hexagonalsystem having the formula Ca_(5x)(PO₄)_(3x),(OH, Cl, F)_(x) with x≧1.

Preparation Methods

Thus, the present invention relates to a method for preparing aninjectable calcium-phosphate bone cement releasing a gem-bisphosphonicderivative comprising the addition of a gem-bisphosphonic compound or acalcium precursor modified with a gem-bisphosphonic derivative, in thesolid phase or in the liquid phase, wherein the gem-bisphosphonicderivative amount is up to 2.5% by weight in respect to the weight ofsolid phase.

The gem-bisphosphonic derivative amount and the way to incorporate it inthe preparation method according to the invention is an essentialfeature in order to provide a cement according to the invention with aninitial setting time suitable for a surgical use, i.e. lower than 1hour, preferably lower than 30 min.

The bisphosphonic acids or salts thereof that may be used asgem-bisphosphonic compounds correspond to the formula:

(OY)(OX)P(O)—CR₁R₂—P(O)(OX)(OY)

wherein X or Y represents, independently of each other, H or an alkalimetal or alkaline-earth metal cation, and any organic or inorganiccation of biological interest.R₁ represents H, OH or a halogen, andR₂ represents a hydrogen or a halogen, an alkyl radical, an aminoalkylradical in which the amino group optionally bears an alkyl substituent,an alkylamino radical, an alkyl radical bearing an aromatic substituentcomprising at least one N atom, an alkyl radical bearing an aromaticthioether group.

When R₁ and/or R₂ represent a halogen, Cl is particularly preferred.

When R₂ is an alkyl radical, alkyls containing from 1 to 6 carbon atomsare preferred.

When R₂ is an aminoalkyl radical, radicals NH₂(CH₂)_(n)— in which n isless than 6 are preferred.

When R₂ is an alkylaminoalkyl radical, the preferred radicals areradicals R′R″N(CH₂)_(m)— in which R′ and R″ represent, independently ofeach other, H or an alkyl radical containing up to 5 carbon atoms, and mis less than 6.

When R is an alkylamino radical, the radicals RCNH— in which RC is acycloalkyl containing from 3 to 7 carbon atoms are preferred.

When R₂ is an alkyl radical bearing an aromatic substituent comprisingat least one N atom, alkyls containing up to 3 carbon atoms and bearingone pyridyl or imidazolyl group are preferred.

When R₂ is an alkyl radical bearing an aromatic thioether group, alkylscontaining up to 3 carbon atoms and bearing a phenylthio group in whichthe phenyl group optionally bears a halogen substituent are preferred.

Among these gem-bisphosphonic compounds, mention may be made of:

-   -   etidronate (R₁—OH, R₂—CH₃),    -   clodronate (R₁—Cl, R₂—Cl),    -   pamidronate (R₁—OH, R₂—CH₂CH₂NH₂),    -   alendronate (R₁—OH, R₂—(CH₂)₃NH₂),    -   risedronate (R₁—OH, R₂—CH₂-3-pyridine),    -   tiludronate (R₁—H, R₂—CH₂—S—C₆H₄—Cl),    -   ibandronate (R₁—OH, R₂—CH₂—CH₂—N(CH₃)pentyl),    -   zoledronate (R₁—OH, R₂—CH₂-imidazole),    -   incadronate (R₁—H, R₂—NH-(cycloheptyl)),    -   olpadronate (R₁—OH, R₂—CH₂—CH₂—N(CH₃)₂),    -   neridronate (R₁—OH, R₂—(CH₂)₅NH₂).

A salt of a bisphosphonic acid may be an organic or mineral salt,preferably an alkali metal or alkaline-earth metal salt.

In a preferred embodiment, a gem-bisphosphonic compound used in themethod according to the invention is selected from the group consistingof etidronate, clodronate, pamidronate, alendronate, risedronate,tiludronate, ibandronate, zoledronate, incadronate, olpadronate, andneridronate.

Three ways may be used to prepare a cement according to the invention:

-   -   The gem-bisphosphonic derivative is dissolved in the cement        liquid phase; or    -   The gem-bisphosphonic derivative is added at the pulverulent        solid phase; or    -   The gem-bisphosphonic derivative is chemically associated to a        calcium precursor and added in the solid or the liquid phase

When the gem-bisphosphonic derivative is dissolved in the cement liquidphase, the gem-bisphosphonic derivative amount is preferably up to 0.3%by weight in respect to the weight of solid phase.

When the gem-bisphosphonic derivative is added at the pulverulent solidphase, the gem-bisphosphonic compound amount is preferably up to 5% byweight in respect to the weight of solid phase.

When the gem-bisphosphonic derivative is chemically associated to acalcium precursor and added in the solid or the liquid phase, thegem-bisphosphonic compound amount is preferably up to 0.15% by weight inrespect to the weight of solid phase.

The chemical association of the gem-bisphosphonic compound may beobtained by adding a gem-bisphosphonic acid or an alkali metal oralkaline-earth metal salt or and any organic or inorganic cation ofbiological interest thereof to a suspension of a precursor phosphocalciccompound, in a solvent preferably an aqueous medium (e.g. ultrapurewater), by stirring the reaction medium at room temperature, and thenrecovering the formed compound by centrifugation. The compound may thenbe purified by washing with ultrapure water, followed by filtering anddrying in air at room temperature.

The calcium precursor is chosen:

-   -   i) from calcium orthophosphates. By way of example, mention may        be made of alpha- or beta-tricalcium phosphate (generally        denoted as α-TCP, β-TCP), CDA, which is a calcium-deficient        hydroxyapatite (obtained, for example, by alkaline hydrolysis of        a calcium hydrogen orthophosphate), hydroxyapatite (HA),        dicalcium phosphate anhydrous (DCPA), CaHPO₄; dicalcium        phosphate dihydrate (DCPD), CaHPO₄.2H₂O, tetracalcium phosphate        (TTCP), Ca₄P₂O₉; amorphous calcium phosphate (ACP),        Ca_(x)(PO₄)y.H₂O; monocalcium phosphate monohydrate (MCPH),        CaH₄(PO₄)₂.H₂O; and,    -   ii) non phosphate calcium salts, e.g. CaCO₃, CaSO₄

“Ultrapure water” means water having a resistivity in the region of 18MΩ cm. The stirring at room temperature is preferably maintained for aperiod of between 1 hour and 72 hours, for example for 48 hours. Thenature of the stirring and the particle size of the calcium precursormay have an effect on the proportion of gem-bisphosphonic compound thatmay be grafted. It is thus preferable, when a given particle size hasbeen selected for the calcium precursor, to adapt the stirring so as notto modify said particle size.

More preferably, the calcium precursor modified with a gem-bisphosphoniccompound and used in the method according to the invention iscalcium-deficient apatite (CDA), α-TCP, DCPD, or CaCO₃.

Cements

The present invention further relates to an injectable apatiticcalcium-phosphate bone cement releasing a gem-bisphosphonic compoundobtainable according to the method of the present invention as describedabove.

Calcium phosphate cements (CPC's) are materials consisting of a liquidphase being water or an aqueous solution and a pulverulent solid phasecontaining one or more solid compounds of calcium and/or phosphate saltsso that if liquid and solid phases are mixed in an appropriate ratiothey form a paste which at room or body temperature sets byprecipitation of one or more other solid compounds, of which at leastone is a calcium phosphate.

CPCs according to the invention are of the CDHA (calcium-deficienthydroxyapatite) type.

The CPC according to the invention is injectable. Indeed, in recentyears, the occurrence of osteoporotic fractures has dramaticallyincreased. Considering the lack of adequate treatment and the increasingnumber of elderly people, this trend is expected to continue.Osteoporotic fractures are often very difficult to repair, because thebone is very weak. It is therefore not possible to insert screws to holdosteosynthesis plates. A way to solve the problem is to inject a CPCinto the osteoporotic bone to reinforce it.

Calcium and/or phosphate compounds useful in the invention as acomponent of the solid phase include hydroxyapatite (HA)Ca₁₀(PO₄)₆(OH)₂; amorphous calcium phosphate (ACP), Ca_(x)(PO₄)y.H₂O;monocalcium phosphate monohydrate (MCPH), CaH₄(PO₄)₂.H₂O; dicalciumphosphate dihydrate (DCPD), CaHPO₄.2H₂O, also called brushite; dicalciumphosphate anhydrous (DCPA), CaHPO₄; precipitated or calcium-deficientapatite (CDA), (Ca,Na)₁₀(PO₄,HPO₄)₆(OH)₂; alpha- or beta-tricalciumphosphate (α-TCP, β-TCP), Ca₃(PO₄)₂; tetracalcium phosphate (TTCP),Ca₄P₂O₉, and calcium carbonate, CaCO₃.

Easily resorbable calcium phosphate compounds are preferred.

A pulverulent solid phase comprising one or more calcium and/orphosphate compounds selected from the group consisting of HA, α-TCP,β-TCP, ACP, MCPH, DCPA, CDA, CaCO₃, and mixtures thereof, is preferred.

According to a particular embodiment, the solid phase can also compriseat least one synthetic polymer or biopolymer (e.g. HPMC).

A pulverulent solid phase comprising α-TCP is more preferred. α-TCP hasthe formula α-Ca₃(PO₄)₂. α-TCP is easily transformed intocalcium-deficient hydroxyapatite (CDA) in aqueous solution. Thisproperty is used to form apatitic CPCs. An α-TCP preferred amount iscomprised between 5% and 100%, more preferably 30% and 80%, and mostpreferably 30% and 70% of the solid phase.

A preferred pulverulent solid phase consists in a mixture of α-TCP,DCPA, CDA and CaCO₃.

Another preferred pulverulent solid phase consists in a mixture ofα-TCP, DCPD, CDA, MCPH, and a biopolymer such as HPMC(hydroxypropylmethylcellulose).

The liquid phase may consist of one or more aqueous solutions containingone or several of the components of Table I, wherein said component maybe chosen among the respective compounds are mixtures thereof shown inTable I.

TABLE I suitable liquid phases Component Compounds Sodium NaF, NaCl,Na₂CO₃, NaHCO₃, Na₂SO₄, Na₂SiO₃, Na ortophosphates Potassium KF, K₂CO₃,K₂SO₄, KCl, K₂SiO₃, K ortophosphates Magnesium MgHPO₄, Mg₃(PO₄)₂•xH₂O,MgF₂, MgCO₃, MgO, CaMg(CO₃)₂, Mg(OH)₂ Zinc Zn₃(PO₄)₂•xH₂O, ZnF₂, ZnCO₃,ZnSO₄, ZnO, Zn(OH)₂, ZnCl₂ Calcium Ca₅(PO₄)₃OH, CaSO₄, CaSO₄•½H₂O,CaSO₄•2H₂O, CaF₂, CaCO₃, CaCl₂ Biopolymers Proteins, peptides,proteoglycans, glycosaminoglycans, carbohydrates Organic acids Citricacid, malonic acid, pyruvic acid, tartaric acid Inorganic acidsPhosphoric acid Synthetic polymers Polylactic acid, polyglycolic acidGrowth factors TGF-β, osteocalcine, GLA proteins

Preferably, the concentrations of aqueous solutions of the compoundsdescribed above as liquid phases are between about 0.1% and about 5% byweight.

A preferred liquid phase consists in a Na₂HPO₄ aqueous solution, aNaH₂PO₄ aqueous solution or a citric acid solution. More preferably, theliquid phase consists in a Na₂HPO₄ aqueous solution. For example, asolution of about 0.5% to about 5% by weight of Na₂HPO₄ in distilledwater, a solution of about 0.5% to about 5% by weight of NaH₂PO₄ indistilled water or a solution of about 0.5% to about 5% by weight ofcitric acid in distilled water can be used.

The pH of the liquid phase should be between about 5 to about 10,preferably between about 5 and about 9, most preferably between about 5and about 7.

Preferably, the liquid phase/solid phase (L/S) ratio is between about0.20 to about 0.9 ml/g, more preferably between about 0.25 to about 0.8ml/g, still preferably between about 0.25 to about 0.45 ml/g, the mostpreferably about 0.30 to about 0.45 ml/g.

Preferably, the liquid phase/solid phase (L/S) ratio is between about0.25 ml/g and about 0.9 ml/g; more preferably between about 0.30 ml/gand about 0.45 ml/g, the liquid phase being an aqueous Na₂HPO₄ solution.

Preferably, the liquid phase/solid phase (L/S) ratio is between about0.25 ml/g and about 0.9 ml/g; more preferably between about 0.30 ml/gand about 0.45 ml/g, the liquid phase being an aqueous NaH₂PO₄ solution.

Preferably, the liquid phase/solid phase (L/S) ratio is between about0.20 ml/g and about 0.8 ml/g; more preferably between about 0.25 ml/gand about 0.30 ml/g, the liquid phase being an aqueous citric acidsolution.

The setting time of a CPC depends on the composition of the powder andliquid components, the powder-to-liquid ratio, proportion of the calciumphosphate components and the particle sizes of the powder components.The setting time of the cement is an important property of the cement.If the setting time is too fast, the surgeon does not have time to usethe cement before it is hard. If the setting time is too long, thesurgeon must wait until he/she can close the wound.

The setting time is usually measured on a moulded sample with a Gillmoreneedle apparatus. This test basically measures when the hydrating cementpaste develops some finite value of resistance to penetration. Itdefines an initial setting time and a final setting time based on thetime at which a needle of particular size and weight either penetrates acement paste sample to a given depth or fails to penetrate a cementpaste sample. The Gillmore needle apparatus consists in two needles witha different diameter and a different weight. The first needle with thebiggest diameter and the lowest weight measures the initial setting timeand the second one with the lowest diameter and the highest weightmeasures the final setting time (C266 ASTM standard).

The initial setting time of the cement according to the invention issuitable for a chirurgical use, i.e. lower than 1 hour, preferably lowerthan about 45 min. Preferably, it is comprised between about 10 min andabout 45 min, more preferably about 15 min and about 40 min, mostpreferably between about 20 min and about 35 min.

The final setting time of the cement according to the invention iscomprised between about 40 min and about 3 h, preferably about 40 minand about 2 h, most preferably between about 40 min and about 1 h.

In a preferred embodiment, the compressive strength of the hardenedcement according to the invention is above about 10 MPa, preferablyabove about 20 MPa.

In order to prevent any extravasation of the cement into the tissuessurrounding bone, it is very important to visualise the cement. Theeasiest way is to increase the radio-opacity of the cement, for exampleby means of contrasting agents. For example, metallic powders oftantalum, titanium or tungsten can be used. It might be preferable touse liquid agents in partially bioresorbable cements, such as iodinecompounds as iopamidol, iohexyl and iotrolan. Preferably, bariumsulphate is used.

Uses

A further object of the invention is the use of an injectable CPCaccording to the invention to fill a bony defect or fracture caused bytrauma, osteoporosis, osteolytic tumours, and articular or dentalprostheses surgeries. This includes a surgery step but injectable CPCsaccording to the invention can get to inaccessible parts of the body andare suited for minimally invasive surgery procedures that are intendedto reduce damage and pain while shortening the time before returning tofunction. This method of treatment comprises the introduction in thebony defect or fracture through a needle of an injectable CPC accordingto the invention.

For example, they can be employed in percutaneous vertebroplasty. Thisconsists of a percutaneous puncture method to stabilize and straightenvertebral collapse of the thoracic and lumbar spinal column, most oftenas a result of osteoporosis.

In the course of osteoporosis, a very painful vertebral collapse canoccur in the region of the thoracic (TSC) and lumbar (LSC) spinal columnas a result of the reduced load-bearing capacity of the skeletal frame.This results in more or less distinct deformation of the vertebrae, andeven in vertebral collapse. Both cases are easily recognizable by x-ray.Even a complete vertebral collapse and distinct deformation of theentire spinal column is possible.

Under local anesthetic, or, if desired, under full narcosis, a thinpuncture needle is inserted to the vertebra, e.g. under x-ray guidance.At a certain point of the vertebra (the so-called pedicel), the bone canbe punctured by the needle without risk. Afterwards, fluid bone cementis injected into the vertebra via the puncture needle; after the cementhardens, the vertebra is stabilized (vertebroplasty). If the vertebra isseverely deformed (e.g. in the case of a wedge-like formation), thecollapsed vertebra is straightened before the cement is injected. Aballoon is hereby inserted into the vertebra via the puncture needle andinflated with fluid under high pressure. Following a successfulstraightening, the balloon is removed and the resulting cavity is filledwith bone cement (balloon-kyphoplasty).

A further object of the invention is the use of an injectable CPCaccording to the invention to fill a tooth defect.

A supplementary object of the invention is a kit for preparing aninjectable calcium-phosphate bone cement releasing a gem-bisphosphonicderivative according to any of claims 8 to 10 comprising agem-bisphosphonic derivative or a calcium precursor modified with agem-bisphosphonic compound, a solid phase and a liquid phase.

The invention will be further illustrated in view of the followingfigures and examples.

FIGURES

FIG. 1: ³¹P VACP MAS NMR spectrum of modified CDA [10.4 wt %alendronate], showing the alendronate component associated to CDA (seeexample 1). The spectra were recorded at a spinning frequency of 12 kHzand a magnetic field of 7.0 T.

FIG. 2: ³¹P MAS spectra of cements (see example 5) after a one weeksetting time. reference=no alendronate added, solid=alendronate powdermixed with the cement solid component, solution=alendronate dissolved inthe cement liquid component, CDA=alendronate chemically associated tothe CDA component

FIG. 3: (upper view) ³¹P single pulse MAS-NMR spectrum of modified α-TCP[4.7 wt % alendronate], (bottom view) ³¹P VACP MAS NMR spectrum ofmodified α-TCP [4.7 wt % alendronate] (see example 2).

FIG. 4: Scanning Electron Microscopy (observation in the backscatteredelectron mode) view of a ewe vertebral body implanted with a 3 g-dose ofunloaded CPC, 12 weeks after implantation.

FIG. 5: Scanning Electron Microscopy (observation in the backscatteredelectron mode) view of a ewe vertebral body implanted with a 3 g-dose ofalendronate-loaded CPC (0.13 wt %), 12 weeks after implantation (seeexample 8).

EXAMPLES Example 1 Preparation of CDA Modified with Alendronate

A suspension of calcium phosphate was prepared by introducing 100 mg ofCDA into 8.75 ml of ultrapure water mixed with 1.25 ml of a 0.02 mol.l⁻¹sodium alendronate aqueous solution. The suspension was placed in a tubemaintained at room temperature, and was stirred with a rotary stirrer at16 rpm for 5 days. The suspension was then centrifuged and the most partof the supernatant was removed. The solid residue was filtered off,washed several times with small portions of ultrapure water, and thendried at room temperature. The resulting solid contained 7.4 wt %alendronate.

Example 2 Preparation of α-TCP Modified with Alendronate

In addition to example 1, the bisphosphonate can also be chemicallyassociated to one of the other components of the solid phase (CaCO₃,DCPA, α-TCP . . . ). For example, in the case of α-TCP, a suspension ofthe calcium phosphate support was prepared by introducing 100 mg ofα-TCP into 8.75 ml of ultrapure water mixed with 1.25 ml of a 0.02mol.l⁻¹ sodium alendronate aqueous solution. The suspension was placedin a tube maintained at room temperature, and was stirred with a rotarystirrer at 16 rpm for 2 days. The suspension was then centrifuged andthe most part of the supernatant was removed. The solid residue wasfiltered off, washed several times with small portions of ultrapurewater, and then dried at room temperature. The resulting solid contained4.7 wt % alendronate.

Example 3 Preparation of DCPD Modified with Alendronate

In addition to example 1, the bisphosphonate can also be chemicallyassociated to one of the other components of the solid phase. Forexample, in the case of DCPD, a suspension of the calcium phosphatesupport was prepared by introducing 100 mg of DCPD into 9 ml ofultrapure water mixed with 1 ml of a 0.02 mol.l⁻¹ sodium alendronateaqueous solution. The suspension was placed in a tube maintained at roomtemperature, and was stirred with a rotary stirrer at 16 rpm for 2 days.The suspension was then centrifuged and the most part of the supernatantwas removed. The solid residue was filtered off, washed several timeswith small portions of ultrapure water, and then dried at roomtemperature. The resulting solid contained 5.3 wt % alendronate.

Example 4 Preparation of CaCO₃ Modified with Alendronate

In addition to example 1, the bisphosphonate can also be chemicallyassociated to one of the other components of the solid phase. Forexample, in the case of CaCO₃, a suspension of the calcium phosphatesupport was prepared by introducing 100 mg of CaCO₃ into 8.5 ml ofultrapure water mixed with 1.5 ml of a 0.02 mol.l⁻¹ sodium alendronateaqueous solution. The suspension was placed in a tube maintained at roomtemperature, and was stirred with a rotary stirrer at 16 rpm for 2 days.The suspension was then centrifuged and the most part of the supernatantwas removed. The solid residue was filtered off, washed several timeswith small portions of ultrapure water, and then dried at roomtemperature. The resulting solid contained 5.0 wt % alendronate.

Example 5 Preparation of an Injectable CPC Releasing Alendronate

The solid phase of the cement consists of alpha-tertiary calciumphosphate α-TCP, CaHPO₄, CaCO₃ and some precipitated hydroxyapatite CDA.

The solid phase composition is the same for all samples:

-   -   62.4 wt % (249.6 mg) α-TCP    -   26.8 wt % (107.2 mg) DCPA (CaHPO₄)    -   8.8 wt % (35.2 mg) CaCO₃    -   2 wt % (8 mg) CDA.

α-TCP was prepared by using an appropriate mixture of CaHPO₄ and CaCO₃,heating it at 1300° C. for at least 6 h and quenching it in air down toroom temperature.

Three ways are used to combine alendronate with the cement samples.

-   -   alendronate is dissolved in the cement liquid phase (up to 1.2        mg in 120 μL see Table IV); or    -   alendronate is added to the solid phase (0.1-10 mg for 400 mg        see Table IV); or    -   alendronate is chemically associated to (i) CDA as prepared in        Example 1 replacing partially the CDA of the solid phase (see        Table II) (ii) α-TCP as prepared in Example 2 replacing        partially the α-TCP of the solid phase (see Table III).

Seven concentrations of alendronate have been used:

-   -   0.100 wt % (0.40 mg)    -   0.060 wt % (0.25 mg)    -   0.025 wt % (0.10 mg)    -   0.25 wt % (1 mg)    -   0.3 wt % (1.2 mg)    -   2.5 wt % (10 mg)    -   3.9 wt % (15.7 mg)

Three liquid phases were chosen to prepare different cementformulations: 2.5% Na₂HPO₄ by weight in water, 2.5% NaH₂PO₄ by weight inwater or 85 mM citric acid in water.

The liquid/powder ratio L/P of cements was taken to be either 0.30 ml/gfor samples prepared with Na₂HPO₄ and NaH₂PO₄ and 0.25 ml/g for samplesprepared with citric acid.

The powders are finely ground during 10 minutes.

Then, the liquid phase is added dropwise and the two phases are mixedwith a spatula or a pestle.

The mixing sets in moulds.

Example 6 Setting Time Assays of the Samples of Example 5

Setting times were determined with Gillmore needles following thestandard ASTM C266-89.

Tables II and III and IV summarize the results.

TABLE II Initial and final setting times with alendronate chemicallyassociated to CDA Liquid m(Alend) ti phase [mg] pH ti [min] tf [min][min] tf [min] T [° C.] Na₂HPO₄ control 8.5 30 75 20 (2.5% by 0.1 8.5 3080 30 70 20 weight)  0.25 8.5 40 90 30-35 80 20 L/S = 0.3 0.5 8.5 40 8535 75 20 NaH₂PO₄ control 5 20 60 21 (2.5% by 0.1 4.5 20 60 20 60 21weight)  0.25 4.5 20 60 20 60 21 L/S = 0.3 0.5 4.5 25 80 20 60 21

TABLE III Initial and final setting times with alendronate chemicallyassociated to α-TCP Liquid ti tf phase m(Alend) [mg] pH [min] [min] T [°C.] NaH₂PO₄ control 5 20 60 21 (2.5% by 0.5 5 30 70 21 weight) obtainedby mixing 4.24 wt % of L/S = 0.3 modified α-TCP [4.7 wt % alendronate]in pure α-TCP^(a) 0.5 5 35 70 21 obtained by using only modified α-TCP[0.2 wt % alendronate]^(b) 1 5 35 70 21 obtained by mixing 8.48 wt % ofmodified α-TCP [4.7 wt % alendronate] in pure α-TCP^(c) 10 5 35 85 21obtained by using only modified α-TCP [4 wt % alendronate]^(d) Note^(a)After one week incubation, a full transformation of α-TCP wasobserved, and the resulting cement showed good mechanical properties.Note ^(b)After one week incubation, the self-setting of the cement wasvery poor, while X-Ray diffraction gave evidence that the transformationof α-TCP was very low. Note ^(c)After one week incubation, a fulltransformation of α-TCP was observed, and the resulting cement showedquite good mechanical properties, although the cement was a little morecrumby than in the case of note a. Note ^(d)After one week incubation,the self-setting of the cement was very poor, while X-Ray diffractiongave evidence that the transformation of α-TCP was very low. From notesb and d, it can be deduced that if the entire α-TCP component ismodified with a bisphosphonate, the self-setting properties of thecement are strongly inhibited.

TABLE IV Initial and final setting times with alendronate dissolved inthe liquid phase or added to the solid phase T = 22° C. Alendronatedissolved in the liquid phase Alendronate added Liquid m(Alend) ti tf tothe solid phase phase [mg] pH [min] [min] pH ti [min] tf [min] Na₂HPO₄control 8.5 25-30 75 8.5 25-30 75 (2.5% by 0.4 6.5-7.0 45 80 8.5 35 95weight)  0.25 6.5-7.0 45 80 8.5 40 90 L/S = 0.3 0.1 6.5-7.0 40 90 8.5 3095 **NaH₂PO₄ control 5 25 65-70 5 25 65-70 (2.5% by 10^(a  ) 5 45 95weight)  1.2^(b) 4.5 65 >100 L/S = 0.3 0.4 4.5 35 75 5 30 80  0.25 4.545 90 5 30 80-85 0.1 4.5 35 60 5 35 80 citric acid^(c) control 2 12 53 212 53 (85 mM) 0.4 2 35 65 2 13 60 L/S = 0.25  0.25 2 20 55 2 15 60 0.1 225 55 2 15 60 Note ^(a)In that case, the amount of α-TCP transformed islow, and after one week incubation, the material is obtained as a chewypaste with poor mechanical properties. Note ^(b)In that case, the amountof α-TCP transformed is low, and after one week incubation, the materialis obtained as a soft paste with very poor mechanical properties. Note^(c)Under incubation conditions, a swelling of the preparation isobserved, and after one week poor mechanical properties were observedfor the material that is brittle and crumby, although a fulltransformation of α-TCP was evidenced by X-ray diffraction.

Example 7 RMN Assays (Concerning the Samples of Example 5)

The cement samples obtained after 7 days incubation were studied usingsolid-state magic angle spinning (MAS) NMR spectrometry. The experimentswere carried out on a Bruker Advance 300 spectrometer, operating at 7.0T (¹H and ³¹P Larmor frequencies of 300 and 121.5 MHz), using 4 mmdouble-resonance and triple-resonance MAS probes.

The ³¹P—{¹H} cross-polarisation (CP) MAS experiments were performedusing a ramped cross polarization with a contact time of 1 ms. ¹Hdecoupling was achieved using the SPINAL64 sequence with a ¹H nutationfrequency of 70 kHz. The recycle delay was set to 2 s. Longitudinalrelaxation times T₁ for ³¹P sites in the modified α-TCP samples weremeasured and found to vary between 10 and 300 s (ν₀(³¹P)=121.5 MHz). The³¹P single pulse spectra were thus obtained by recording a single scanafter a delay of 600 s.

Example 8 Preparation of a Second Type of Injectable CPC ReleasingAlendronate

The solid phase of the cement consists of alpha-tertiary calciumphosphate α-TCP, DCPD, MCPH, HPMC and some precipitated hydroxyapatiteCDA.

The solid phase composition is the same for all samples:

-   -   78 wt % (7.8 g) α-TCP    -   5 wt % (0.5 g) DCPD (CaHPO₄.2H₂O)    -   5 wt % (0.5 g) MCPH (Ca(H₂PO₄)₂.H₂O)    -   10 wt % (1 g) CDA.    -   2 wt % (0.2 g) HPMC (hydroxypropylmethylcellulose).

α-TCP was prepared by using an appropriate mixture of CaHPO₄ and CaCO₃,heating it at 1300° C. for at least 6 h and quenching it in air down toroom temperature.

Three ways are used to combine alendronate with the cement samples.

-   -   alendronate is dissolved in the cement liquid phase (up to 40 mg        in 5 mL see Table V)    -   alendronate is added to the solid phase (13.3-40 mg for 10 g see        Table V)    -   alendronate is chemically associated to (i) CDA as prepared in        Example 1 replacing partially the CDA of the solid phase (see        Table VI) (ii) α-TCP as prepared in Example 2 replacing        partially the α-TCP of the solid phase (see Table VII) (iii)        DCPD as prepared in Example 3 replacing partially the DCPD of        the solid phase (see Table VIII)

Three concentrations of alendronate have been used:

-   -   0.133 wt % (13.3 mg)    -   0.266 wt % (26.6 mg)    -   0.4 wt % (40.0 mg)

The liquid phase chosen to prepare different cement formulations was 5%Na₂HPO₄ by weight in water. The liquid/powder ratio L/P of cements wastaken to be 0.50 ml/g.

The powders are finely ground during 30 minutes.

Then, the liquid phase is added dropwise and the two phases are mixedwith a spatula or a pestle.

The mixing sets in moulds.

Example 9 Setting Time Assays Related to Example 8

The properties of the cements were studied using Vickersmicroindentation (maximal compressive strength), powder X-raydiffraction and ³¹P solid state NMR (transformation ratio of α-TCP toCDA), and texture analyses (evaluation of the initial setting time). Thelatter method consists in measuring the compression force necessary toextrude the cement dough (initial setting time=the time to reach a forcevalue>25 Newton) versus time.

Tables V, VI, VII and VIII summarize the results.

TABLE V Setting and mechanical properties of cements with alendronatedissolved in the liquid phase or added to the solid phase MaximalTransformation m(Alend) compressive Initial setting of α-TCP to [mg]strength [MPa] time [min] CDA Alendronate dissolved in the liquid phase0 (control) 11 ± 1 15 high 13.3 18 ± 3 65 high 26.6 19 ± 1 >100 high40^(b )  20 ± 3 >>250 high Alendronate added to the solid phase 0(control) 11 ± 1 15 high 13.3 11 ± 1 45 high 26.6 Notmeasurable^(a) >100 high 40^(b )  Not measurable^(a) >>100 Very highNote a: in that case, after two days incubation, the material isobtained as a brittle and crumby material, leading to non-reproducibledata. Note b: the presence of alendronate is detected on ³¹P-¹H VACP NMRspectra, as a broad signal at ca. 18 ppm, very similar to that presentin FIG. 1, thus suggesting that the bisphosphonate is chemisorbed on thesurface of the CDA resulting from the transformation of the α-TCPcomponent.

TABLE VI Setting and mechanical properties of cements with alendronatechemically associated to CDA Alendronate associated to CDA MaximalTransformation m(Alend) compressive Initial setting of α-TCP to [mg]strength [MPa] time [min] CDA 0 (control) 11 ± 1 15 high 13.3 16 ± 2 40high 26.6 19 ± 2 90-100 high 40^(a )  18 ± 2 >>90 high Note a: Thepresence of alendronate is detected on ³¹P-¹H VACP NMR spectra, as abroad signal at ca. 18 ppm, very similar to that present in FIG. 1, thussuggesting that the bisphosphonate is chemisorbed on the surface of theCDA resulting from the transformation of the α-TCP component.

TABLE VII Setting and mechanical properties of cements with alendronatechemically associated to α-TCP. Alendronate associated to α-TCP InitialMaximal setting Transformation compressive time of α-TCP to m(Alend)[mg] strength [MPa] [min] CDA 0 (control) 11 ± 1 15 high 13.3 13 ± 1 40high 26.6 12 ± 1 >120 high 40 12 ± 1 >>120 high 13.3, obtained by mixing14 ± 1 17 high 6 wt % of modified α-TCP [2.85 wt % alendronate] in pureα-TCP 26.6, obtained by mixing 14 ± 1 30 high 12 wt % of modified α- TCP[2.85 wt % alendronate] in pure α- TCP 40, obtained by mixing 18 wt 15 ±1 75 high % of modified α-TCP [2.85 wt % alendronate] in pure α-TCP

TABLE VIII Setting and mechanical properties of cements with alendronatechemically associated to DCPD. Alendronate associated to DCPD Maximalcompressive Initial setting Transformation m(Alend) [mg] strength [MPa]time [min] of α-TCP to CDA 0 (control) 11 ± 1 15 high 13.3 22 ± 3 60high 26.6 20 ± 1 >>90 high 40 20 ± 2 >>90 high

For the different cases corresponding to Tables V-VIII (0.133 wt %alendronate relative to the solid phase), cement blocks obtained after 2hours incubation were immerged in a 0.9 wt % NaCl aqueous solution at37° C. for 5 days. The blocks were then dried and cut, before SEM(Scanning Electron microscopy) observations. In all cases, homogeneouslydispersed macropores (20 to 100 μm) were observed, resulting from thedegradation of the HPMC component.

Example 10 In Vivo Assays in Ewes Related to Example 8

Six 10-years-old ewes are used for this experiment. The animals had freeaccess to normal diet. The animals were randomly implanted withalendronate-loaded or unloaded CPC. 3 g-doses of alendronate-loaded CPCwere prepared according to example 8 with 4 mg of alendronate chemicallyassociated to CDA (see example 1). Each ewe received 3×3 g-doses (sharedinside 3 vertebral bodies) of either alendronate-loaded or unloaded CPC.Animals were sacrificed 3 months after implantation. Each implantedvertebral body was analysed using:

-   -   1. Scanning Electron Microscopy (observation in the        backscattered electron mode)    -   2. Micro-CT scan (histomorphometric measurements).

Auto-induced macroporosity, direct interface with bone and significantsurface osteoconduction were observed on SEM images in both CPC andalendronate-loaded CPC (FIGS. 4 and 5). Significant implant resorptionand volumic osteconduction were observed for alendronate-loaded CPCimplants (FIG. 5). Comparative histomophometric analysis of thetrabecular bone structures surrounding CPC and alendronate-loaded CPCimplants demonstrated (N=18, p<0.05) clearly that the bone architectureis reinforced by alendronate-loaded CPC implants (see table below).

Bone Trabecular Trabecular Volume (%) space (μm) number (μm⁻¹) CPC 17.7± 1.5 494.9 ± 5.8 0.95 ± 0.07 CPC + 28.4 ± 2.7 420.7 ± 8.0 1.53 ± 0.22alendronate

1. A method for preparing an injectable calcium-phosphate bone cementreleasing a gem-bisphosphonic compound, said method comprising theaddition of a bisphosphonic acid or a salt thereof or a calcium solidprecursor modified with a bisphosphonic acid or a salt thereof, in thecement solid phase or in the cement liquid phase, said calcium solidprecursor being chosen from calcium orthophosphates, and non phosphatecalcium salts, such as CaCO₃ or CaSO₄
 2. The method according to claim1, wherein the amount of the bisphosphonic acid or salt thereof is up to2.5% by weight in respect with the weight of solid phase.
 3. The methodaccording to claim 1, wherein the bisphosphonic acid or salt thereof isdissolved in the cement liquid phase and the amount of the bisphosphonicacid or salt thereof is up to 0.3% by weight in respect with the weightof solid phase.
 4. The method according to claim 1, wherein thebisphosphonic acid or salt thereof is added in the cement solid phaseand the amount of bisphosphonic acid or salt thereof is up to 3.5% byweight in respect with the weight of solid phase.
 5. The methodaccording to claim 1, wherein a calcium solid precursor modified with abisphosphonic acid or salt thereof is added in the cement solid phaseand the amount of the bisphosphonic acid or a salt thereof is up to0.15% by weight in respect with the weight of solid phase.
 6. The methodaccording to claim 1, wherein the bisphosphonic acid or salt thereof isselected in the group consisting of etidronate, clodronate, pamidronate,alendronate, risedronate, tiludronate, ibandronate, zoledronate,incadronate, olpadronate, and neridronate.
 7. The method according toclaim 6, wherein said bisphosphonic acid or salt thereof is alendronate.8. The method according to claim 1, wherein said calcium solid precursoris chosen from the group consisting of: calcium-deficient apatite (CDA),α-TCP, DCPD, and CaCO₃.
 9. The method according to claim 1, wherein saidsolid phase comprises one or more calcium and/or phosphate compoundsselected from the group consisting of HA, α-TCP, β-TCP, ACP, MCPH, DCPA,CDA, CaCO₃ and mixtures thereof.
 10. The method according to claim 1,wherein the solid phase also comprises at least one synthetic polymer orbiopolymer.
 11. The method according to claim 1, wherein said liquidphase consists in a Na₂HPO₄ aqueous solution, a NaH₂PO₄ aqueous solutionor a citric acid aqueous solution.
 12. The method according to claim 1,wherein said liquid phase consists in a NaH₂PO₄ aqueous solution. 13.The method according to claim 1, wherein the pH of the liquid phase isbetween 5 to 10, preferably between 5 and 9, most preferably between 5and
 7. 14. The method according to claim 1, wherein the liquidphase/solid phase (L/S) ratio is between 0.20 to 0.9 ml/g, morepreferably between 0.25 to 0.8 ml/g, still preferably between 0.25 to0.45 ml/g, the most preferably 0.30 to 0.45 ml/g.
 15. An injectableapatitic calcium-phosphate cement obtainable according to the method ofclaim
 1. 16. An injectable apatitic calcium phosphate cement accordingto claim 15, with a compressive strength above 10 MPa.
 17. An injectableapatitic calcium phosphate cement according to claim 15, includingfurther a contrasting agent, preferably barium sulphate.
 18. The use ofan injectable apatitic CPC according to claim 15, for the manufacture ofa medicament to fill a bony defect or fracture.
 19. The use according toclaim 18, wherein said bony defect is associated with osteoporosis. 20.The use of an injectable apatitic CPC according to claim 15, for themanufacture of a medicament to fill a tooth defect.
 21. A kit forpreparing an injectable calcium-phosphate bone cement releasing abisphosphonic acid or salt thereof according to claim 15 comprising abisphosphonic acid or salt thereof or a calcium solid precursor modifiedwith a bisphosphonic acid or salt thereof, a solid phase and a liquidphase, said calcium solid precursor being chosen from calciumorthophosphates, and non phosphate calcium salts, such as CaCO₃ orCaSO₄.